We will isolate anti-HIV neutralizing antibodies (NAbs) from a yeast-displayed library of human nonimmune scFv antibodies. Developed at Pacific Northwest National Laboratory, the PNNL scFv library is a high diversity library which has been used by numerous investigators to generate high-quality, high-affinity scFv antibodies to a myriad of protein antigens. The yeast display platform is also ideal for engineering scFv antibodies by affinity maturation through random mutagenesis. We will perform selections on the PNNL scFv library using HIV glycoprotein 140 (gp140), gp120- and gp41-derived peptides, and intact virions. We will introduce a novel method to secrete soluble biotinylated scFv for direct screening for neutralization of HIV. NAbs isolated from this library will then be subjected to affinity maturation to improve the breadth and potency of neutralization. Affinity matured NAbs, which vary in overall affinity as well as in their kinetic on- and off-rates, will be compared to their parental NAb for neutralization of a diverse panel of HIV isolates. This proposal will provide insights into two key questions: 1) can anti-HIV neutralizing scFv antibodies be isolated from a nonimmune library of human scFv antibodies;and 2) can the breath and potency of neutralization by these scFv be improved upon by improving their on-rates, off-rates, and affinities. These studies will identify new anti-HIV neutralizing antibodies and their epitopes as well as provide useful data for selection and engineering of NAbs isolated from any source.